Molecular Studies on the Alcohol Dehydrogenase Gene of Drosophila melanogaster

Year: 1984 DOI: 10.7907/skx9-0t33
In this thesis, I describe the isolation of the alcohol dehydrogenase (Adh) gene of Drosophila melaogaster and some preliminary biochemical characterizations of the gene and its expression. The isolation of the Adh gene was accomplished by screening a bacteriophage λ library containing inserts of Drosophila DNA with eDNA probe made from size selected mRNA. One clone which showed hybridization in the initial scr•en was shown to contain Adh sequences by virtue of its lack of hybridization to Adh deficiency DNA, in situ hybridization, translation of ADH protein by mRNA selected by hybridization to the clone, and by partial DNA sequence analysis. Using the clone, approximately 35 kb of the Adh chromosomal region was isolated. This region was found to be composed largely of single-copy sequences, showed limited polymorphism between strains, and encoded only one RNA transcript prevalent in larvae and adults - the Adh mRNA. Two intervening sequences within the Adh coding region were demonstrated by S1 nuclease mapping.

In order to identify sequences important in Adh expression, the cloned Adh gene was transformed into the Drosophila germ line by utilizing the hybrid dysgenesis P element vector of Spradling and Rubin. The correct developmental expression of the Adh gene was retained by the transformed gene, even though it had integrated into many locations. These results delimit the sequences and chromoscmal enviroiiilent necessary for correct developmental expression of the Adh gene. In addition, the 'transient' expression of cloned DNA in larvae and adults directly grown from injected embryos was investigated. In most instances, ADH activity was found only in tissues that normally express ADH, although low level of activity was observed in some cells which do not normally produce detectable levels of Adh. Together, these results form the basis for assay systems that may be combined with in vitro mutagenesis in order to determine in detail which sequences are necessary for correct developmental expression of the Adh gene.